Method of treating colic with trihydroxyacetophenones



United States Patent 3,137,621 METHOD OF TREATWG COLIC WITHTRIHYDRQXYACETDPHENQNES Louis Laion, Paris, France, assignor to SocieteAnonyme dite: Orsymonde, Paris, France, a French company No Drawing.Qriginal application Feb. 20, 1961, Ser.

No. 90,169. Divided and this application Nov. 29,

1961, Ser. No. 155,790

6 Claims. (Cl. 167-55) The present invention relates by the formula:

to substances represented In which it will be seen a crotonisation (or-.-COCH=CH forming) reaction is carried out. This application is adivision of our copending application Serial No. 90,169, filed Feb. 20,1961.

Trihydroxyacetophenone, used as the starting material, can be obtainedby treating phloroglycinol with acetonitrile in the presence ofanhydrous hydrogen chloride and zinc chloride; the hydrochloride ofcetimine thus formed is then hydrolysed by heating with water.

Trihydroxyacetophenone has the form of a pale yellow powder, slightlysoluble in water, soluble in ether, very soluble in alcohol andinsoluble in chloroform. Its melting point is 2l6218 C.

There are given below, by way of illustration, nonlimiting examples ofthe process according to the invention, giving new substances:

EXAMPLE 1 Benzylidene-trihydroxyacetophenone 10.6 g. (0.1 mole) offreshly distilled benzaldehyde were added to 16.8 g. (0.1 mole) oftrihydroxyacetoe phenone. The paste thus obtained was triturated with 2x 3 ccs. hydrochloric acid and allowed to stand for 48 hours; itincreased in weight and 15 ccs. acetic acid were Patented June 16, 1964and very soluble in chloroform.

The yield of the reaction is Analysis: Calculated (percent), C=70.31,H=4.68. Found (percent), 0:69.57, H=4.93.

This substance has some antispasmodic activity; also, it has ahypotensive eifect and causes a decrease in intestinal peristalsis andcholeresis in dogs at a dose of 50 mg./kg.

EXAMPLE 2 Ortho-chlorobenzylidene-trihydroxyacetophenone JH t The samemethod was employed as described in EX- ample 1, starting with 16.8 g.(0.1 mole) of trihydroxyacetophenone and 14.0 g. (0.1 mole) ofortho-chlorobenzaldehyde.

27.4 g. of a yellow powder were obtained, which is insoluble in water,slightly soluble in ether, chloroform and alcohol and more soluble inthese materials in the hot.

The heated powder becomes brown around 185 C. but without melting evenat 325 C.

The yield of the reaction is Analysis: Calculated (percent), C=61.96,H=3.75. Found (percent), 0:60.91, H=4.01.

This substance has an antispasmodic activity of the same order as theproduct of Example 1. Its toxicity is relatively low; its DL byintraperitoneal administration in mice is 1.60 mg./kg.

In the dog anaesthetised with chloralose, the substance in a dose of 30mg./kg. administered intravenously produces a slow hypotensionpersisting for 30 mins., together with an ephemeral decrease inperistalsis and a slight decrease in respiratory amplitude; a completecessation of choleresis was noted at this dosage.

7 EXAMPLE 3 Ortho-hydroxy benzylia'ene-zrihydroxyacetophenone The samemethod was used as in Exam le 1, starting with 16.8 g. (0.1 mole) oftrihydroxyacetophenone and 12.2 g. (0.1 mole) of salicaldehyde. 20 g. ofa product soluble in acetic acid were obtained at the end of thepreparation; it could be separated as by dilution of the acetic solutionwith water. It was purified by redissolving in 95% alcohol andprecipitating with water.

A reddish-orange powder is obtained in this manner, which is insolublein water, very soluble in acetone and alcohol, and soluble in aceticacid.

Its melting point is 172174 C.

The yield of the reaction is 75%.

Analysis: Calculated (percent), C=66.17, H=4.41. Found (percent),C=65.84, H=4.60.

This compound has an antispasmodic activity of the same order as that ofthe compounds of Examples 1 and 2.

3 EXAMPLE 4 Vanillidelze-trihydrxyacetophenone The method described inExample 1 was employed, starting with 16.8 g. (0.1 mole) oftrihydroxyacetophenone and 15.2 g. (0.1 mole) of vanillin.

10 cos. of a 1:5 solution of hydrochloric acid were used and a productwas obtained which was dried and slowly washed with water and then withchloroform; it was dried and 26 g. of a yellow powder were obtained,which is insoluble in water, soluble in benzene and chloroform in thehot and very soluble in acetone and alcohol.

Its melting point is 220 C.

The yield of the reaction is 89%.

Analysis: Calculated (percent), C=63.15, H=5.26. Found (percent),C=62.87, H'=5.45.

The latter substance has very good antispasmodic properties and can beused in the treatment of hepatic disorders and of nephritic colics.

EXAMPLE 5 Parahydroxybenzylidene-trihydr0xyacetophenone I OH -For thispreparation, 16.8 g. trihydroxyacetophenone and 12.2 g.parahydroxybenzaldehyde (0.1 mole) were used and the method described inExample 1 was employed; 26,6 g. of product were obtained, namely 96% ofthe theoretical. This product has the form of a reddish-yellow powder,insoluble in water and soluble in alcohol, particularly hot; on heating,the powder turns brown around 210 C. and decomposes around 270 C.

Analysis: Calculated (percent), 0:66.17, H=4.41. Found (percent),0:67.51, H'=4.96.

EXAMPLE 6 M eta-hydroxybenzylidene-trihydroxyacetophenone OH on i Thegeneral method was employed, with 16.8 g. trihydroxyacetophenone and12.2 g. of metahydroxybenzaldehyde (0.1 mole). This produced 26 g. (or ayield of 95%) of product, which has the form of an orange powder,insoluble in water and soluble in acetone and alcohol; on heating, thepowder turns brown around 230 C. and decomposes without melting at 272C.

Analysis: Calculated (percent), C=66.17, H=4.41. Found (percent),C=65.20, H'=4.65.

The compounds of Examples 5 and 6 have a very marked vasodilatoryaction, which is particularly so in relation to coronary sufierers.

EXAMPLE 7 Parachlorobenzylidene-trihydroxyacetophenone The generalmethod was also employed, with 7.8 g. trihydroxyacetophenone and 14 g.parachlorobenzaldehyde (0.8 mole) and 27 g. (or a yield of 95%) ofproduct were obtained, which has the form of a light yellow powder,

insoluble in Water, slightly soluble in benzene, soluble in alcohol andvery soluble in ether and chloroform. 0n heating, the powder changescolour around 190 C. without melting. Its analysis gives:

Calculated (percent), C=6l.96, H=3.75. Found (percent), C=60.15,H'=3.97.

This compound has a certain antispasmodic activity, similar to that ofits isomer in Example 2; however, the para derivative of the presentexample has a lower toxicity than its ortho isomer; the DL byintraperitoneal administration in mice is 3.80 rug/kg.

Also in a dog anaesthetised with chloralose, the compound in a dose of19 mg./ kg. administered intravenously exerts a rapid, large, butephemeral hypotension; it does not cause any cessation of choleresis.

The products obtained according to the invention are particularlyinteresting in therapeutics, where their antispasmodic and/or coronaryvasodilative properties can be employed.

The compound of Example 4, namely vanillidenetrihydroxyacetophenone, isparticularly interesting and a pharmacodynamic study carried out on thiscompound is given below.

The toxicity of this product has been studied, in the first place.

Female mice of the Webster strain have been used for this, weighingbetween 16 and 25 g. and maintained throughout the test in separatecages; these mice were fed and watered as desired.

The first tests carried out on 48 mice by intraperitoneal administrationand it was observed that the average toxic dose of the product is 0.355g./kg.; the toxicity was studied gastrically on 60 mice and it wasobserved after 7 days that the average toxic dose is 5.10 g./ kg.

The antispasmodic action was then studied on isolated rat duodenummaintained alive by the Magnus method. For this the duodenum wasmaintained by immersion in oxygenated Tyrode liquid at a temperature of32 C. and spasmogenic agents such as barium chloride and acetylcholinewere used.

It was observed on the one hand that the spasm produced by 5 mg. bariumchloride was diminished by 46% on adding 15 mg. of the product to theTyrode bath ccs.).

It was also observed on the other hand that the spasm produced by 10gamma acetylcholine was diminished by 80% on the addition of 0.5 g. ofthe product to the Tyrode bath (60 ccs.); such an effect is identicalwith that obtained with papaverine. 4

- Further studies were made to find whether the product likewise exertsan antispasmodic effect in situ and dogs anaesthetised with chloraloseand subjected to artificial respiration were used for this; theperistaltic movements were recorded by the method of a balloon insertedin the duodenum and connected to a Marcy capsule. Intestinal spasm wasproduced by intravenous injection of 3 mg./kg. barium chloride and itwas observed that doses of 60 mg./ kg. of the product were slightlyactive.

The cardiovascular effect of the product was also studied on dogsanaesthetised with chloralose and it was observed that doses of theorder of 60 mg./kg. cause a drop in the carotid pressure of about 33%.

It was also studied Whether such a hypotensive effect was the result ofa cardiac action. The action of the product on the heart of the rabbitin situ was studied for this purpose. v It was observed that such anaction was not higher than the hypotensive effect of the solvent used,namely propylene glycol diluted to 25%. A dose of 7 mg./kg. of thisproduct did not cause any cardiac depressive action.

Doses of 200 to 300 mg./kg. of the product were also used, which cause adiminution of the carotid pressure without having any cardiachypotensive action.

The action of the product on the coronary output'was then studied, usingthe Langendorfi method on isolated rabbit heart; it was observed that adose of gamma, the product caused a decrease in the coronary output.

The action of the product was also studied on isolated guinea-pib uretermaintained alive in an analogous manner to that of the Magnus methodused for the duodenum.

The pendular rhythmic movements of the ureter which resemble cardiacpulses which are observed on the heart of a mouse perfused by the methodof Straub are accelerated and there can even be obtained a spasm withthe aid of barium chloride which is subsequently added to the Tyrodebath. It was observed also that the product clearly exerts anantispasmodic action on the isolated ureter.

The action of the product on diuresis was also studied, utilizing themethod of Lichfitz on a rat. It was noted that, as regards aqueousdiuresis, the product causes an increase in the diuretic activity fordoses of 1 to 1.5 g./kg.; however, the product does not modify theresorbtion of tubular chlorine and does not modify the pH at the dosesused.

The choleretic action of the studied.

In the first place, the action was studied on chronic choleresisutilising the Ruteford technique on four dogs; a dose of 5 mg./kg.caused a decrease followed by an increase of choleresis; a dose of 10mg./kg. caused an increase of choleresis; a dose of 10 mg./kg. caused anincrease of choleresis in one case, and in another case, a diminutionfollowed by a temporary increase of the biliary output, itself followedby a considerable decrease.

In the second test, the action on choleresis was studied in dogs havingan acute choledoch fistula and it was observed that a dose of 10 mg./kg.caused a diminution in choleresis of 28%.

It was thus shown that the product has a very low toxicity administeredbucally, that it exerts a musculotropic and neurotropic antispasmodicaction, that it also exerts a hypotensive action and a diuretic actionand finally that it diminishes choleresis.

While the compounds have not yet been established as effective in humantherapy a first clinical study has enabled it to be shown thatvanillidene-trihydroxyacetophenone has a spectacular antispasmodicactivity, particularly in the treatment of nephritic colic and hepaticcolic; it can be employed in doses of 0.10 g. absorbed 3 to 6 times perday.

This compound can be put up in the form of cachets, tablets, capsules orsuppositories and glucose can be utilized as an excipient in associationwith 0.10 g. of the compound, in quantities from 0.10 to 0.60 g. It canalso me made up in an injectable form being associated if desired withthe product known as Pluronic.

The compound of Example 5, namelyparahydroxybenzylidene-trihydroxyacetophenone, is particularlyinteresting; it can be used in therapeutics because of its coronaryvasodilatory action in the treatment of coronari tis and diseases duetoperipheral vaso-constriction. human medicine, a dose of 0.10 g. of thecompound absorbed 3 to 4 times per day can be used.

A pharmacodynamic study of this compound is given below:

The acute toxicity of the compound by intraperitoneal administration hasbeen determined on 56 female mice of the Webster strain, having weightsranging from 16 to 25 g. The animals were maintained throughout thewhole of the period of observation (3 days) in individual cages. Theproduct was administered to groups of 8 mice in doses ranging from 50 to750 mg./ kg. body weight.

The average mortal toxic dose, 131. hasbeen determined by the graphicmethod of Tainter and Miller and appears to be 225 mg./kg.

The effect of'the compound has also been studied on isolated organs: ratduodenum, guinea-pig ureter and coronary rabbit artery.

The rat duodenum was maintained alive by the Magnus product was finally6 method; oxygenated Tyrode liquid was maintained at a temperature of 32C. The spasmogenic effect was produced either by means of a neurogenicagent, acetylcholine, or a musculotropic agent, barium chloride.

As regards the musculotropic effect, the spasm was induced by theaddition to the Tyrode liquid of 0.16 mg. of barium chloride per 1 cc.of liquid; the compound in a dose of 1.60 mg./cc. alleviated the spasm.

As regards the neurotropic eflect, the spasmogenic agent,acetylocholine, was utilised in a dose of 1.5 gammas/cc.

The compound is 4000 times less active than atropine sulphate used as areference.

The guinea-pig ureter was maintained alive in a special Locke liquid(deprived of alkaline phosphates and magnesium chloride) at atemperature of 37 C. As the spasmogenic agent, barium chloride was usedin a dose of 0.10 mg/cc. of liquid. No spasm was produced, but therhythm and amplitude of the pendular movements increased.

A dose of 0.5 mg./cc. of compound does not modify the rhythm of periodicmovements; on the contrary, it increased them. On the other hand, a doseof 0.83 mg./ cc. diminished the amplitude and then produced a cessationof the automatic movements; thus the effect on the ureter is relativelyweak.

On the coronary rabbit arteries, technique was utilised. A Van Dyke andHastings solution was added to the Tyrode liquid, carbogen wassubstituted for oxygen; the vasodilative effect was produced byperfusing the heart with a Van Dyke and Hastings solution includingbarium chloride which produced a persistent and stable vasoconstriction.

Tests have been carried out on four hearts at concentrations varyingfrom 1x10 to 1X 10-. It was always noted that the compound exerts avasodilatory action which, compared with that of papaverine on 3 hearts,appeared to be 10 times greater. Also the elfect of the compound is morelasting than that of papaverine.

The effect of the compound on organs in situ was also studied; rabbitheart, cat heart, dog duodenum and finally the carotid pressure of thedog.

On a rabbit anaethetised with urethane and subjected to artificialrespiration, the standard suspension method was utilised, in which thecompound is either in a gummy suspension or in solution in propyleneglycol; simultaneously the carotid pressure was recorded by means of "avery sensitive mercury manometer.

The tests were carried out on 3 rabbits.

In a dose of 10 mg./ kg. of gummy suspension, the product caused a dropin carotid pressure of 20% which was fugitive, followed by a slow andprogressive decrease of about 60%, at a maximum after 8 minutes, andthen a progressive increase without return to the normal; Recording ofthe amplitudes of the auricle and ventricle showed a sharp decrease. Adose of 20 mg./kg., which roduced a persistent and progressive drop inthe pressure, exerts a decrease of the amplitude of the ventricle andthen on that at the auricle, ending in cardiac failure 1 hour afterinjection.

In a dose of 1 trig/kg. dissolved in propylene glycol, the compound hasno efiect on the pressure, no effect on the ventricle and causes aslight decrease in the auricular amplitude. A dose of 2 mg./kg; whichproduces a drop in the pressure of 25%, also produces a slight andfugitive decrease in the auricular and ventricular amplitudes. Finally,a dose of 5 mg./kg., which produces a drop in pressure of is accompaniedby a slightdecrease in the auriculo-ventricular amplitude. It may bementioned that on control animals, propylene glycol in the sameconcentrationproduces a less significant drop in pressure and decreasein amplitude. p

A cat anaesthetised with nembutal was experimented upon by using thesame technique of recording the movethe Langendorff ments of the auricleand ventricle by the suspension method and of the carotid pressure bymeans of a mercury manometer.

The test was carried out on 3 cats, which appeared to be more sensitivethan the rabbit. The compound in a dose of mg./kg. injected slowly instabilised aqueous suspension at a concentration of 5 X" produced acollapse of the arterial pressure and a decrease in the amplitude of theauricle and of the ventricle with auriculoventricular dissosiation;these effects were mortal.

On a second cat, a dose of l mg./kg. administered in aqueous solution ata concentration of 1X 10- produced a drop of 68% of the arterialpressure and a decrease of 50% in the amplitudes of the auricle and theventricle. These phenomena are reversible.

The renewed administration of 1 mg./kg. effected 90 minutes after thefirst injection produced a lesser effect; the drop of carotid pressurewas only 50%, the decrease in the amplitude of the auricle was only 30%and that of the ventricle was 17%. It seems that the repetition of theeffects gives rise immediately to a tachyphylaxic phenomenon.

The tachyphylaxia is confirmed by the fact that subsequentadministration of 2 mg./kg. only diminishes the pressure by 36% and doesnot modify the auriculoventricular action; in addition, injections of 5mg./kg. and 10 mg./kg. appear then to be ineffective.

On a third cat, the hypotensive and temporary cardiac depressive effectsof 1 mg./kg. of the compound were confirmed.

The vascular effect of the compound was confirmed on 5 mongrel dogsnormotensively anaesthetised with chloralose and on which the carotidpressure was recorded by the standard method by use of a Ludwigmanometer.

A dose of 1 mg./kg. exerts a drop in carotid pressure which variesaccording to the animal from 34 to 80%. A dose of 5 mg./kg. is likewisehypotensive and non-toxic. A dose of 13 mg./kg. administeredintravenously appears to be lethal.

On the same dogs, the respiratory effect was recorded by connecting atracheal canulla to a Marey capsule.

Doses of 1 and 5 mg./kg. produced a temporary decrease of therespiratory amplitude with acceleration of the rhythm.

The action on duodenal peristalsis of a dog anaesthetised withchloralose was studied by the standard method of a balloon inserted inthe duodenum and connected to a Marey capsule.

A dose of 1 mg./ kg. increased the peristalsis. At a dose of 5 mg./kg.,however, a decrease of peristalsis was noted which continued untilparalysis set in, but this was temporary.

The modifying action of a compound on the biliary flux in the dog hasbeen studied according to two different methods, namely, acute choledochfistula and chronic choledoch fistula.

On a dog anaesthetised with chloralose, a polyethylene probe wasintroduced into the choledoch duct, after ligature of the cystic duct.When the biliary flux was stabilised, which was generally observed 2hours after anaesthetising with chloralose, the biliary output wasrecorded by means of an electric drop-counter, connected to anelectromagnetic stylus inscribing upon a kymograph.

Even a dose of 1 mg./kg. decreased the biliary flux. A dose of 5 mg./kg. on another dog provoked an analogous effect. a

For chronic choledoch fistula, the choledoch duct was catheterised in afirst stage under general anaesthesia, after having ligatured the cysticduct which eliminates vesicular bile. The choledoch duct wascatheterised by means of a polyethylene probe of 1 mm. diameter whichwas con nected externally after suture to the abdominal wall in thesub-cutaneous region. Each day the animal received 50 cc. of beef bileincluded in a rich paste of chopped raw meat.

After 1 or 2 weeks, the animal was taken for physiological testing.First, the base choleresis was determined by measuring the volume ofbile eliminated every 15 minutes until it become constant. Then thecompound was injected intravenously in doses of 2 mg./ kg. to 3 animals;the compound was a gum-stabilised 1% suspension.

In three cases, there was obtained a diminution of choleresis varyingfrom 20 to 65 but this diminution in choleresis is not stable.

The diuretic effect was studied on 3 dogs anaesthetised with chloralose,after ureteral catheterisation by means of a polyethylene probe.Recording of the drops was effected by means of an electric drop-counterutilizing the same device as that mentioned above.

In a dose of 1 mg./kg. in 1 case, an increase of 20% in the diuresis wasnoted, in a second case an increase of 20 to 60% in the diuresis whichpersisted for 1 hour; in a third case, a decrease of diuresis wasobtained, on the other hand; a dose of 5 mg./kg. employed on 2 dogsalways caused diminution of diuresis.

The pharmacodynamic study was completed by determining the modifyingaction with regard to two peripheral poisons, adrenalin andacetylcholine, the effect of which was recorded on a dog normotensivelyanaesthetised with chloralose.

Doses of the compound varying between 1 and 5 mg./ kg. did not modifyeither the hypertensive effect of adrenalin nor the hypotensive effectof acetylcholine.

The compound does not have any effect on the central nervous system; itis neither analgesic nor ataraxic in a. strong dose, but does exert ahypothermisic effect on mice.

The compound of Example 6, namelymetahydroxybenzylidene-trihydroxyacetophenone, likewise has a very clearvasodilatory action.

A pharmacodynamic study of this compound is given below.

The acute toxicity of the compound was determined on 40 mice of theWebster strain, weighing from 15 to 20 g. An aqueous suspension of thecompound stabilised by gum at 5% was utilised which was injected to 5groups of 8 mice.

The DL is 300 mg./kg. The limits of toxicity in 19/20 of the casesvaried between 244 and 367 mg./kg.

The effect of the compound has been studied on the intestinal system.

On isolated rat duodenum, the standard Magnus technique was used, inwhich the organ was maintained at a temperature of +32. C.

The antispasmodic musculotropic and neurotropic effects weresuccessively recorded.

The musculotropic spasmogenic agent was barium. chloride dissolved 'in adose of 0.16 mg./ cc. in oxygenated Tyrode.

The compound in a dose of 1.6 mg./kg. exerts an action comparable withthat of the compound of Example 5.

The antispasmodic neutropic effect was studied on the same preparationutilising acetylcholine in a dose of 0.16 gamma/ cc. as the spasmogenicagent.

In a dose of 1.6 mg./cc., the compound decreases the spasm produced byacetylcholine by more than 50%, the compound having an effect 18,000times less than that o atropine sulphate.

The effect of the compound on peristalsis was studied by the standardmethod of a rubber balloon in contact with a Marey capsule by means of aprobe.

Experiments have been carried out on a dog anaesthetised with chloralosewhich minimises the inhibition of peristalsis by anaesthesia.Experiments were carried out on 3 mongrel dogs using different dosesadministered intravenously.

Even a dose of l mglkg. decreases the tonus and peristaltic movements; adose of 10 mg./ kg. is even more active and a dose of 16 mg./kg. is evenmore active and a dose of 16 mg./ kg. completely retards peristalsisduring jtrically. The measurement of urinary volume allowed the horal 9a period of 13 minutes and it is necessary to wait a further 30 minutesto observe a return to normal.

Another test in vivo has been carried out to determine the efiect of thecompound on the parasympathetic system, in particular on salivarysecretion.

For this purpose, the secretoryefrect of mecholyl on e Harderian glandsin the rat has been studied (Hamburger, Cahen and Tvede). Although inall the control animals the haematoporphyric secretion(chromodacryorrhea) appeared after intraperitoneal injection ofmecholyl, previous injection of the compound in a dose of 0.150 gjkg.administered intraperitoneally prevented this action in 50% of the rats'20 to 30 minutes after injection and lasted for more than 60 minutes. Ahigher dose of 250 mg./kg. showed activity on 75% of the animals. Thiseifect is similar to that obtained in another group of animals of 0.03rag/kg. of atropine sulphate administered introperitoneally.

The diuretic effect was studied using two different techniques on twospeciesof animals; the rat and the dog.

The diuretic efiect was determined on the rat in accordance with themethod of Lipschitz, Hadidian and Kerpsar, modified by R. Cahen (Jour.Amer. Pharm. Ass., Scient. Ed. 1947, 36, pp. 139-144). It consists indetermining the volume, the urinary excretion and the re absorption ofchlorine.

White rats were utilised, of the male sex and Wistar strain, weighingfrom 130 to 160 g. The animals were deprived of food and water for 18hours and wer grouped in sizes in metabolic cages having the bottomfurnished with a screen, for permitting flow of urine into a graduatedreceiver. All the animals were rehydrated before the experiment bygastric administration of physiological serum.

Before beginning the experiment, the bladder was emptied by excretionand either the compound or a reference was then administered bucally.Determination of the diuretic eifect was then carried out from thehourly urinary elimination and the volume eliminated was measured bymeans of a graduated receiver. The experiment was carried out on a groupof six animals for each of the doses studied. It was carried out incomparison with two groups of controls which had only receivedgastrically administered physiological serum. Finally, the tests werecarried out in comparison with a reference material, acetazolamide.

The compound was administered gastrically in doses of 500 mg. and 1g./kg., the reference being utilised in doses of mg. and mg./kg., alsoadministered gasurinary output to be established, i.e., the volumeexcreted per hour. It also allowed establishment of the urinaryelimination and diuretic activity.

Determination of the 'urinmy volume allowed determination in the firstplace of the urinary excretion, which is the ratio of the volume ofurine eliminated and the volume of physiological serum administered tothe rat. 7

This allows the diuretic activity then to be established as calculatedfrom the ratio of the urinary excretion of the animals subjected totesting to that of the controls receiving only physiological serum onthe same day.

The chloruria was determined on samples taken every two hours. Themethod of determination utilised is that of Charpentier-Volhard. Theresults were expressed in microequivalents per per minute.

It appears that the compound in a dose of 500 mg./kg. exerts a diureticactivity comparable to that of 15 or 30 mg./kg. of acetazolamide. A doseof 1 g./kg. of the compound exerts a lesser effect, but still greaterthan that of the controls.

The deterrrunatiou of the horal urinary output allows it to beestablished that the maximum eifect of the com- The compound in a doseof 0.50 g./kg. slightly increases the chloruria during the first hour;on the other hand, confirming the standard information, acetazolamideproduces no hyperchloruria.

In addition, the elfect of the compound on the motility of the ureterhas been studied. An isolated guinea-pig ureter was used for thispurpose, maintained alive by a technique analogous to that of Magnus, ina special Locke liquid deprived of magnesium salts and phosphates.

The contraction stimulating agent was barium chloride in a dose of 0.10rug/cc. which on an organ substantially deprived of peristalsis producesperiodic pendular contractions.

The addition of the compounds to the organ previously treated withbarium chloride, even in a doseof 0.75 mg./cc., only slightly modifiesthe effect.

The effect of the compound has also been studied on hepato-biliarysecretion in the dog.

On an anirnal anaesthetised with chloralose, the biliary flux wasrecorded after the choleretic effect of the chloralose has dissipated.

Under anaesthesia, the choledoch duct was catheterised after ligature ofthe cystic duct. In general, choleresis is stabilised 2 hours after theonset of anaesthesia. The base choleresis was observed, the product wasinjected and the biliary flux was measured every 15 minutes. The biliaryoutput is measured by means of an electric dropcounter in relation to astylus inscribing on a kymograph.

The study was carried out on 3 dogs to which the compound: wasadministered intravenously in a dose of 1 mg./kg. In this case,adiminution of choleresis was observed which was at a maximum after 30minutes but persisted for more than one hour.

On another dog, a dose of 10-mg./kg. also diminished choleresis.Finally, a third dog received 13 mg./kg. intravenously and the biliaryflux was likewise decreased.

These experiments have been conformed by determining the biliary fiux onanimals with a'chronic choledoch fistula.

For this purpose, after having effected fistulation of the choledochduct and ligature of the cystic duct under anaesthesia, the animals werereleased and the bile which had flowed into a probe supportedexternallyby suture to the abdominal wall was recovered after 1 or 2Weeks. The volume was measured by means of a graduated receiver. Thebiliary constituents were also determined, namely bilirubin, cholic acidand cholesterol.

Experiments have been carried out on a dog which had receivedintravenously 2 mg./l g. of the compound in a 1% suspension atdiminution of choleresis of 26% was observed during /2v hour and theeffect was prolonged during hour; It was observed that the concentrationof the biliary constituents remained the same but, because of thediminutionof biliary volume, there was a diminution in the volume of thebilirub'in, cholic acid and cholesterol. 1

The effect of the compound on the respiratory system was studied in 3dogs anaesthetised with'chloralose in which the variation in therespiratory amplitude and volume were recorded by means of a Marcycapsule in relation to a trachael canulla. Doses of 1-5 and 10 mg./kg.diminished the respiratory rhythm but slightly increased the amplitude.

The effect of the compound on the "cardiac system has i been studied inthe cat anaesthetised with nembutal. Contractions of the auricle andventricle were recorded independently by the suspension method.

In adose of. 5 mgjkg. administered intravenously, the

compound produces a slight decrease'of the auricular and ventricularamplitudej however, the elfect is reversed and disappears after 8minutes. It is interested to note pound is observed at the end of 1 to-2hours; that of that this test gave rise to a'tachyphylaxic phenomenon.The first injection diminished the amplitude by 70%, but the secondgiven 10 minutes after the first only reduced the amplitude by 42%. i

It may be mentioned that this weak and reversible effect does notprohibit the compound from being used in human medicine.

The vascular effect of the compound has been studied on the dog and thecat normotensively by utilizing the standard method of recording thecarotid pressure by means of a Ludwig manometer.

Experiments carried out on 2 cats have shown that a dose of mg./ kg.produces a diminution of the pressure of 24%, a dose of mg./kg. producesa drop of 63%; injection repeated 1 hour after the first dose produced alesser eifect; the hypotension being only 36%.

On 3 dogs, the hypotensive effect of the compound has been confirmed;even a dose of 1 mg/kg. produced a weak hypotension (16%). On the otherhand, a dose of 13 rug/kg. produced a considerable drop in the pressure.

On a dog anaesthetised with chloralose, the modifying etfect of thecompound on the vascular effect of adrenalin and acetylcholine was alsostudied. Even at a dose of 13 mg./kg. which produced a considerablehypotension, the compound did not modify either the hypertensive efiectof adrenalin or the hypotensive effect of acetylcholine.

On isolated rabbit ear perfused by the Pissenski method, thevasodilative elfect of the compound was first confirmed and then themodifying effect was studied in relation to a poison such as bariumchloride.

The standard method of Pissenski was used. The flow output was measuredwith precision by means of an electric drop-counter in relation to arecording stylus inscribing on a kymograph. The experiment was carriedout at laboratory temperature, but the perfusion liquid was a specialLocke solution deprived of magnesium and phosphate, which thus allowedincorporation of barium chlo ride in a dose of 1.5 mg./cc.

In 4 tests, it was established that the compound increases the perfusionoutput of a preparation which had thus been subjected to thevasoconstrictive effect of barium chloride. A dose of 1.5 gammas/cc. ofthe compound exerts a vasodilatory eifect of 30 to 48%.

Study of the compound was completed by examination of the effect on thecoronary vessels of the rabbit.

The method utilised was that of Langendorff involving perfusion of therabbit heart. This method has been rendered more sensitive and constant,(a) by subsituting, for the Ringer liquid, a Van Dyke and Hastingssolution through which a carbogen stream is bubbled, (b) by producing avasoconstriction by dissolution in the Van Dyke and Hastings liquid ofbarium chloride in a concentration of 1.5 X 10- The tests were carriedout on 4 hearts. In all cases, it was apparent that the compound exerteda very pronounced coronary vasodilatory action. At a concentration of 1X10**, the effect could be compared with that of papaverine hydrochlorideat a concentration of 2X10- The compound is approximately 5 times moreactive than papaverine.

From the qualitative point of view, it is interesting to note that theeffect of the compound is more prolonged than that of papaverine.

The two' compounds of Examples 5 and 6 can be utilised in therapeuticsin forms similar to those mentioned for the compound of Example 4. I

12 What is claimed is: 1. A method of treating animals suffering fromcolic which comprises administering to such animals a pharmaceuticalcomposition the essential active ingredient of which is anantispasrnodic agent of the formula:

wherein R, R and R are each selected from the group consisting ofhydrogen atoms, halogen atoms, hydroxy and lower alkoxy, in the amountof 0.10 to 1 gram in a pharmaceutical carrier.

2. A method of treating animals sulfering from me phritic colic whichcomprises administering to such animals a pharmaceutical composition theessential active ingredient of which is an antispasmodic agent of theformula:

wherein R, R and R are each selected from the group consisting ofhydrogen atoms, halogen atoms, hydroxy and lower alkoxy, in the amountof 0.10 to 1 gram in a pharmaceutical carrier.

3. A method of treating animals sufiering from hepatic colic whichcomprises administering to such animals a pharmaceutical composition theessential active ingredient of which is an antispasmodic agent of theformula:

ceutical composition the essential active ingredient of which isp-hydroxybenzylidene-trihydroxyacetophenone in the amount of 0.20 to0.60 gram in a pharmaceutical carrier.

6. A method of treating animals suffering from colic which comprisesadministering to such animals a pharmaceutical composition the essentialactive ingredient of which ism-hydroXybenzylidene-trihydroxyacetophenone in the amount of 0.10 to 1gram in a pharmaceutical carrier.

References Cited in the file of this patent Inagaki: Chem. Abst. vol.51, 1957, page 3833f. Chem. Abs., Formanek, vol. 53, 1959, page 15380.

1. A METHOD OF TREATING ANIMALS SUFFERING FROM COLIC WHICH COMPRISESADMINISTERING TO SUCH ANIMALS A PHARMACEUTICAL COMPOSITION THE ESSENTIALACTIVE INGREDIENT OF WHICH IS AN ANTISPASMODIC AGENT OF THE FORMULA: